Test for predicting neutralization of asparaginase activity

ABSTRACT

Method of in vitro measurement of the presence of factors that are able to neutralize asparaginase activity in a sample of blood, plasma, serum or derived medium that may contain asparaginase neutralizing factors, obtained from a patient, comprising mixing of said sample with asparaginase, incubation of said mixture, then measurement of the residual asparaginase activity in the mixture and determination or quantification of the presence of said neutralizing factors. Method for predicting the efficacy of a treatment with asparaginase.

The invention relates to a test for diagnosing a patient's capacity forresponding to treatment with the therapeutic enzyme asparaginase. Itnotably relates to a test for predicting the efficacy of a treatmentusing asparaginase in a given patient.

L-asparaginase is an essential component of the chemotherapy protocolsthat have been used for more than 30 years for the treatment of acutelymphoblastic leukaemias. Its mechanism of action is based on thehydrolysis of the plasma amino acid asparagine, an essential element fortumoral growth of lymphoblasts. In contrast to normal cells, cancerouslymphoblastic cells are unable to produce their asparagine themselves,and are dependent on extracellular sources. Treatment with asparaginasedeprives them of this essential constituent and thus brings about theirdeath.

The enzyme produced from microorganisms is currently marketed in threeforms: the first two are derived from bacterial sources Escherichia coliand Erwinia chrysanthemi, the third is obtained by covalent-bondcoupling of polyethylene glycol to the native asparaginase ofEscherichia coli (PEG-asparaginase).

Despite its considerable antileukaemic efficacy, treatment withasparaginase is associated with a certain number of complicationsconnected with the immunogenicity of the enzyme. These complications maybe reflected in clinical manifestations or in silent inactivation.

Hypersensitivity reactions, with severity varying from moderate allergicreaction to anaphylactic shock, have been reported by many authors (Wanget al., Journal of Immunological Methods 239 (200) 75-83). Developmentof reactions of this type, observed with the three forms ofasparaginase, generally leads to discontinuation of the treatment forfear of a more severe reaction.

In addition, asparaginase causes the appearance of circulatingantibodies that possess neutralizing properties, reflected in anincrease in clearance of the enzyme by the reticuloendothelial systemand a decrease in its therapeutic efficacy (Müller H. J., Boos J. Crit.Rev Oncol/hematol 1998; (28):97-113). These antibodies have beenobserved with the three forms of the enzyme (E. coli, Erwinia andPEG-asparaginase), and in this instance the therapeutic objective ofasparaginase, which is to achieve rapid and complete depletion of plasmaasparagine for an extended period, is not attained.

This inactivation of the enzyme by neutralizing factors, mainlyantibodies, present in the patients' serum is not accompanied byclinical signs, it is silent for the clinician.

The inventors have identified that there is therefore a considerableneed for clinicians to have at their disposal a test that is quick andis easy to use, for predicting the presence of asparaginase neutralizingfactors, mainly antibodies, present in patients' serum. This test wouldmake it possible to adjust the dose of enzyme administered or to replacethe asparaginase used with another form of asparaginase that is notsensitive, or is less sensitive, to these neutralizing factors.

Several possibilities were considered for monitoring asparaginaseactivity in a patient:

-   -   Assay of plasma L-asparagine    -   Assay of plasma L-asparaginase activity    -   Assay of anti-asparaginase antibodies

The level of plasma asparagine is the main biochemical parameterreflecting the therapeutic effect that is desired with asparaginase: arapid, complete and long-lasting depletion of asparagine. Severalmethods have consequently been described for its assay. Mostly they arebased on combining a stage of separation of the constituents of thesample to be assayed by high-performance liquid chromatography andfluorometric detection or quantification by mass spectrometry. Thesemethods are tedious, they require trained personnel, and their cost andthe time taken are incompatible with routine clinical use.

More recently, Verma et al. described a method for rapid assay ofasparagine based on the co-immobilization of L-asparaginase and acoloured indicator on various substrates (nitrocellulose membrane,silicone gel or beads of calcium alginate). When one of these supportsis brought into contact with a sample of a patient's serum, theimmobilized L-asparaginase will degrade the asparagine present. Theproduction of ammonium following this hydrolysis reaction will lead to acolour change of the phenol red indicator. Although this method appearsto meet the requirements of simplicity and speed of use, the authors donot supply data by which it can be validated, and there are still doubtsabout its accuracy.

Apart from the absence of validated methods that can be used easily andquickly, assay of plasma L-asparagine is limited by the followingconsiderations. In vivo, the degradation of asparagine by the action ofasparaginase is counterbalanced by the physiological production ofasparagine, whereas in vitro, the catalytic effect of the enzyme onasparagine will persist. As a direct consequence, when a serum sample istaken from a patient treated with asparaginase, the presence of aresidual amount of enzyme will lead to a bias in measurement of theasparagine level, which will be “falsely” lower than the physiologicallevel (Boos et al., European journal of cancer (1996) 32, 1544-1550).This interference in the analytical procedure results in absence ofcorrelation with the physiological asparaginase activity in the patienttested.

Several methods for assay of plasma L-asparaginase have been described,and that used most often is based on incubation of the serum containingL-asparaginase in a buffer containing L-asparagine, then after stoppingthe reaction, determination of the ammonium produced using Nesslerreagent. Orsonneau et al. proposed a quicker and more accurate automatedvariant, with which patients treated with L-asparaginase can bemonitored (J. L. Orsonneau et al. Ann Biol Clin 2004, 62: 568-72). Thismethod is based on the action of glutamate dehydrogenase, which uses theammonium produced during the hydrolysis of L-asparagine byL-asparaginase to convert α-ketoglutarate to glutamic acid.Glutamate dehydrogenase α-ketoglutarate+NH₄ ⁺+NADPH→L-glutamate+NADP+H₂O

In this reaction, the amount of NADPH oxidized in the course of thereaction is equivalent to the amount of ammonia contained in the sampleand can be determined by measuring the decrease in optical density. Thekinetics of appearance of ammonium can thus be monitored and theactivity of the L-asparaginase can be calculated. Although this methodmakes it possible to monitor the L-asparaginase activity in a patient,it does not give any predictive information regarding the neutralizationof this enzyme by factors present in the serum.

Wang et al. developed a standardized ELISA test for quantifyinganti-asparaginase IgGs in plasma samples from patients (B. Wang et al.,Journal of Immunological Methods 239 (2000) 75-83). This test was usedwithin the scope of a clinical study for measuring the concentration ofanti-asparaginase antibodies present in the serum of patients with acutelymphoblastic leukaemia, treated with L-asparaginase and who did or didnot develop an allergic reaction (M. H. Woo et al., Leukemia (1998) 12,1527-1533). The authors were able to show that the median concentrationof anti-asparaginase antibodies was higher in the patients who developedan allergic reaction regardless of whether the measurement is carriedout before or after said reaction has occurred. They conclude that thereis a benefit in clinical practice of using such a test for predictingthe future development of an allergic reaction.

Nevertheless, the predictive value of such a test can be questioned; theranges of variation of the concentration of anti-asparaginase antibodiesmeasured before the development of an allergic reaction overlap, theyare respectively:

-   -   from 0.001 to 0.375 unit of OD for the patients who developed an        allergic reaction subsequently;    -   from 0.004 to 0.064 unit of OD for the patients who did not        develop a subsequent allergic reaction

Moreover, measurement of the concentration of antibodies does not giveany information regarding the pharmacological activity of asparaginasein the patient and its possible neutralization by factors present in theserum.

E. H. Panosyan et al., J. Pediatr. Hematol. Oncol. 2004, 26, 4: 217-226investigated the anti-asparaginase antibody and Asparaginase enzymaticactivity in the sera of patients. The authors describe an ex vivoneutralization assay conducted using patient's serum specimens as asource of anti-asparaginase antibodies. The serum specimens wereincubated with native or PEG-asparaginase antigen solutions and theremaining asparaginase enzymatic activity was measured. The authorsfinally recommended the standard monitoring of serum anti-asparaginaseantibodies in clinical settings.

The aim of the present invention is therefore to propose a novelapproach that makes it possible to overcome the drawbacks of the priorart and to know at a given moment whether a patient has factors that canneutralize asparaginase activity. The method must have the advantage ofbeing completely predictive, i.e. it must not require a stage ofadministration of the enzyme to the patient to be diagnosed. It mustreflect the patient's capacity for responding to any form ofasparaginase. Thus, the patient may be a patient who has to be treatedfor the first time using the enzyme, or a patient who has been treatedor is currently being treated with asparaginase. It is possible to testfor the presence of factors that can neutralize the activity of theenzyme used for previous or current treatment, which makes it possibleto know whether the treatment with this enzyme can be resumed orcontinued, and optionally adjusted. It is also possible to detect thepresence of factors that can neutralize the activity of the enzymeconsidered for the treatment of said patient, which makes it possible tovalidate or rule out the use of a particular enzyme.

This knowledge will provide the practitioner with far more pertinentguidance, than with the methods of the prior art, on the form oftreatment (posology, dosage regimen) or on the choice of enzyme or itsform of administration. Either the neutralizing factors are absent orare present at a low enough level for treatment by means of the testenzyme to be possible, optionally with strengthening of the dosage or ofthe dosage regimen. Or the neutralizing factors are present at a levelthat is too high for such a treatment to be continued or initiated, andthen the invention makes it possible to test and/or recommend analternative solution using another form of the enzyme or a form that isless sensitive or is not sensitive to the neutralizing factors, such asthe enzyme included in a biovector.

The invention thus aims to propose an in vitro method of determinationof the presence of factors that neutralize asparaginase activity in ablood sample from a patient.

It also aims to propose a method for in vitro determination of apatient's capacity for responding (i) positively to treatment with anasparaginase or (ii) of not responding to it or (iii) only respondingincompletely.

It also aims to propose a method for predicting the efficacy of atreatment using asparaginase or the fact that this enzyme will notimmediately be the object of substantial inactivation of its activity byneutralizing factors.

The invention therefore relates to a method for predicting whether anasparaginase can be active in a patient, wherein one measures in vitrothe presence of factors that neutralize asparaginase activity in asample of blood, plasma, serum or derived medium that may contain saidneutralizing factors, obtained from said patient.

The invention relates to a method of in vitro measurement of thepresence of factors that neutralize asparaginase activity in a sample ofblood, plasma, serum or derived medium that may contain saidneutralizing factors, obtained from a patient, comprising mixing of saidsample with an asparaginase, incubation of said mixture, thenmeasurement of the residual activity of this asparaginase in themixture, which reflects and makes it possible to determine and quantifythe presence of neutralizing factors in the sample and therefore in vivoin the patient. The method makes it possible to diagnose, qualitativelyand quantitatively, the presence of asparaginase neutralizing factors ina patient. By neutralizing factors, it is intended not onlyanti-asparaginase antibodies, but also any other factor that may inhibitasparaginase or its enzymatic activity, for example proteases such ashuman asparaginyl endopeptidase.

The invention further relates to a method for predicting whether a givenasparaginase can be active in a given patient. This method comprises thedetermination in vitro of a patient's capacity for responding totreatment with an asparaginase, in which a sample of blood, plasma,serum or derived medium that may contain factors that neutralizeasparaginase activity, obtained from said patient, is submitted to themethod comprising mixing of said sample with said asparaginase,incubation of said mixture, then measurement of the residual enzymaticactivity of said asparaginase in the mixture, which reflects and makesit possible to determine and quantify the presence of neutralizingfactors in the sample and therefore in vivo in the patient, that areable to neutralize asparaginase activity, and therefore the patient'scapacity to respond to treatment with this enzyme. The inventiontherefore offers a method for testing the efficacy of an asparaginasefor a particular patient.

Depending on the presence or absence of neutralizing factors, andoptionally on their level, the process and the method make it possibleto determine whether the patient is likely to (i) respond positively totreatment with asparaginase or (ii) not respond to it or (iii) onlyrespond incompletely.

The sample can come from a patient currently being treated with anasparaginase or from a patient who has been treated with anasparaginase.

The sample can also come from a patient who has never been treated withan asparaginase or with this asparaginase.

The asparaginase used in the test can be the one that is being or wasused in the treatment carried out on the patient.

It can also be an enzyme from a different source that we wish to test inthe patient to predict its efficacy. It is also possible to test variousenzymes simultaneously or successively to determine the most suitabletreatment for the patient.

The invention therefore provides a method for predicting the efficacy ofa treatment with asparaginase or for predicting the fact that thisenzyme will not immediately undergo substantial inactivation byneutralizing factors.

The invention applies to all forms of asparaginase, for exampleL-asparaginase. Without being limited to them, we may mention the nativeenzymes obtained from any bacterial source, for example L-asparaginaseproduced by E. coli, the enzyme produced by Erwinia, mutated enzymes ormodified enzymes, for example the pegylated enzymes (PEG-asparaginase).The enzyme can also be of natural, synthetic or recombinant origin. Itcan be free or included in a biovector, for example in erythrocytes.

The residual activity of the asparaginase in the mixture can be measuredby adding, to said mixture, asparagine, preferably L-asparagine and areagent system that is able to detect the enzymatic degradation ofasparagine by active asparaginase.

According to an advantageous configuration, the method comprises thefollowing stages:

-   (a) incubation of the sample with a known amount of asparaginase;-   (b) incubation of the aforesaid mixture with a known amount of    asparagine, preferably in an amount causing saturation relative to    the amount of asparaginase;-   (c) incubation of the aforesaid mixture with the reagent system that    can provide assay of the residual enzymatic activity;-   (d) qualitative or quantitative evaluation of the loss or retention    of enzymatic activity, which correlates with the presence or the    content in the sample, of factors that neutralize asparaginase    activity.

The incubation in stage (a) takes notably from 1 to 60 min. The enzymecontent is notably from 0.1 to 5 IU/ml.

It may be useful and advantageous to inactivate or remove any trace ofactive enzyme in the test sample. Thus, according to one characteristic,before stage (a), a stage (a₀) of removal or inactivation of anyasparaginase present in the sample is carried out.

As a variant, before stage (a) it is possible to carry out a stage (a₀)of measurement of the baseline asparaginase content of the sample, whichmakes it possible either to verify that said activity is zero ornegligible, or to subtract said activity from that measured by themethod.

As a variant, knowing the half-life of the enzyme administered to thepatient before the test, we can wait the necessary time between the lastadministration and taking the blood sample.

According to a particular embodiment, stage (a) is followed by a stage(a₁) of removal of the antibody-asparaginase immune complexes. Saidremoval can be effected easily by centrifugation, so that the mixtureinvolved in stage (b) is the supernatant. Preferably, centrifugation iscarried out at 3000-25000 g for between 1 and 30 min at the specifiedspeed.

According to one characteristic, the reagent system is sensitive to theappearance of the ammonium ion resulting from the enzymatic degradationof asparagine by asparaginase. Thus, the presence of neutralizingfactors can be determined or measured by carrying out a reaction thatconsumes the ammonium ion quantitatively. Said consumption of theammonium ion can be followed, advantageously quantitatively, bymeasurement of the decrease in optical density (absorbance) of themixture. Notably the following reactions are carried out:asparaginase+asparagine→aspartic acid+NH₄ ⁺  (1)α-ketoglutarate+NH₄ ⁺+NADPH+glutamate dehydrogenase(catalyst)→glutamate+NADP⁺+H₂O.  (2)

Incubation with asparagine is preferably carried out for 2 to 60 minwith a saturating amount of asparagine (relative to the amount of enzymeintroduced), notably from 10 to 50 mg/ml.

Incubation with the reagent system notably takes from 3 to 20 min.

The invention therefore provides a method for determining whether thepatient is likely to (i) respond positively to treatment with anasparaginase or (ii) not respond to it or (iii) only respondincompletely. It therefore makes it possible to confirm or to rule outthe possibility of using treatment with the test enzyme, or to make adecision to modify the dosage regimen or to treat the patient using adifferent asparaginase, notably of a form encapsulated in a biovector.

The invention therefore also relates to a method of treatment of anasparaginase-sensitive pathology in a patient, comprising:

(A) application of the method of determination in vitro of a patient'scapacity for responding to treatment with a given form of asparaginase(notably free or modified form), in which a sample of blood, plasma,serum or derived medium that may contain asparaginase neutralizingfactors, obtained from said patient, is submitted to the methodcomprising mixing of said sample with said form of asparaginase,incubation of said mixture, then measurement of the residual activity ofasparaginase in the mixture and determination of said neutralizingactivity and of the patient's capacity to respond (i) positively totreatment with this form of asparaginase or (ii) not respond to it or(iii) only respond incompletely, and(B) treatment of the pathology by means of this asparaginase in case (i)or by means of another form of asparaginase in other cases.

According to a preferred embodiment, the other form of asparaginase isasparaginase encapsulated or included in a biovector, notablyencapsulated in erythrocytes. Notably they are erythrocytes produced bylysis and resealing, for example according to the teaching of Frenchpatent application No. 0408667.

The pathologies that may benefit from this method include in particularleukaemias, for example acute lymphoblastic leukaemias. We may alsomention, without being limited thereto, solid tumours (WO2007/103290),notably pancreatic cancer and ovarian cancer.

The invention will now be described in more detail by means of examples,which illustrate but do not limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described with regard to examples usingthe Figures as follows:

FIG. 1 is a graph showing effect of patient serum matrix onL-asparaginase activity in which seven fixed L-asparaginaseconcentrations (ranging from 0 to 800 IU/L were measured in three humansera and buffer controls.

FIG. 2 is a graph showing effect of patient serum matrix onL-asparaginase activity measurement with distribution of activitymeasured for 52 human sera mixed with L-asparaginase at a finalconcentration of 500 IU/L.

FIG. 3 is a graph showing L-asparaginase activity inhibition versusanti-asparaginase IgG concentration.

FIG. 4 is a graph showing L-asparaginase activity inhibition versusanti-asparaginase IgG low concentration.

FIG. 5 is a graph showing L-asparaginase activity inhibition versusL-asparaginase concentration.

FIG. 6 is a graph showing % of inhibition of L-asparaginase activity in57 human sera from 17 patients treated with L-asparaginase.

EXAMPLES Example 1 Immunization of a Rabbit with L-Asparaginase

A few milliliters of serum are taken from the rabbit before the firstimmunization so as to have a pre-immune serum. Then the rabbit isinjected 4 times with 500 μg of L-asparaginase (Kidrolase®, OPI-EUSALimonest France). Sera are taken between the first and secondimmunization and between the second and third immunization. Finally,after the last immunization, the total serum is recovered and stored at−20° C. The final serum is characterized according to its total proteinconcentration (Biuret method) and its total immunoglobulinconcentration.

Example 2 Purification of Rabbit Total IgGs

Purification of the rabbit total IgGs from the final serum, containinganti-asparaginase IgGs, is carried out with the Kit pure 1A from Sigma(# PURE1A). Briefly, 2 ml of serum is clarified by centrifugation orfiltration on a 0.45 μm filter before purification of the IgGs. Then 4ml of “binding buffer” is added to the 2 ml of clarified serum. Themixture is passed, then eluted from the column following the protocolrecommended by Sigma. So that they can be injected in the animal, thetotal IgGs are centrifuged in a filtration column with a threshold of10000 Dalton in order to replace the elution buffer with PBS.

Example 3a Measurement of the Inhibition of an Intermediate Serum on theEnzymatic Activity of L-Asparaginase (Assay of the Mixture)

Assay of the L-asparaginase was carried out according to the protocolpublished in: Orsonneau et al., “Automatic kinetic assay of plasmaL-asparaginase activity in therapeutic monitoring of acute lymphoblasticleukaemias”, Ann Biol Clin, 62: 568-572.

An intermediate serum (obtained between the first and secondimmunization) was used first, for elaborating measurement of theinhibition of enzymatic activity. A concentration of 2 IU/ml ofL-asparaginase is used. The enzyme is pre-incubated for 15 minutes at37° C. with several dilutions of serum, then the enzymatic activity ismeasured in the mixture. The results are presented in Table 1:

TABLE 1 L-asparaginase Tube L-asparaginase Serum in the mixture ResidualNo. added (IU/ml) dilution (IU/ml) activity, % 1 2 — 1.96 100 2 2 1.110.72 36.43 3 2 1/2  0.78 39.63 4 2 1/4  0.85 43.04 5 2 1/16 1.18 60.16 62 1/32 1.29 65.91 7 2  1/128 1.64 83.74

Table 1 summarizes the measurements of residual enzymatic activities ofL-asparaginase in the mixtures (tubes 2 to 7). The rabbit serum inhibitsthe enzymatic activity of L-asparaginase: the greater the dilution ofthe serum, the weaker the inhibition.

Tube 1 constitutes a control, showing that incubation of the enzymealone at 37° C. for 15 minutes does not affect its enzymatic activity.

Example 3b Measurement of the Inhibition of an Intermediate Serum on theEnzymatic Activity of L-Asparaginase (Assay of a Supernatant)

An intermediate serum (obtained between the first and secondimmunization) was used.

In order to simulate phagocytosis of the antigen-antibody complexes bythe reticuloendothelial system, the L-asparaginase/serum mixture isincubated for 15 minutes at 37° C., then centrifuged for 10 minutes at17500 g at 4° C. in order to remove the immune complexes. The enzymaticactivity is assayed in the supernatant. The assay results are presentedin Table 2:

TABLE 2 L-asparaginase Residual Tube L-asparaginase Serum in supernatantactivity in No. added (IU/ml) dilution (IU/ml) supernatant (%) 1 2 —1.91 95.63 2 2 1.11 0.21 10.39 3 2 1/2  0.14 6.77 4 2 1/4  0.05 2.72 5 21/16 0.03 1.53 6 2 1/32 0.02 0.98 7 2  1/128 0.02 1.00 control 2pre-immune 1.82 90.81

A control, replacing the serum with the pre-immune serum, was added soas to test the specificity of the interaction between L-asparaginase andthe anti-asparaginase antibodies present in the serum. This demonstratesthat more than 90% of the enzyme is not involved in interaction withnonspecific antibodies.

The antibodies present in the serum interacted with all of the enzymefor the dilutions 1/4, 1/16, 1/32 and 1/128.

Example 4 Measurement of Inhibition of the Rabbit Total IgGs from theEnzymatic Activity of L-Asparaginase

The same experiment as that presented in Example 3b was carried out withthe rabbit total IgGs and a concentration of 1.25 IU/ml ofL-asparaginase. The results are presented in Table 3:

TABLE 3 L-asparaginase Residual Tube L-asparaginase Dilution of insupernatant activity in No. added (IU/ml) the IgGs (IU/ml) supernatant(%) control 1.25 — 1.19 94.99 1 1.25 — 1.26 100.00 2 — 1/8 0.02 1.38 31.25 1/8 0.02 1.52 4 1.25  1/16 0.01 0.40 5 1.25  1/32 0.01 0.62 6 1.25 1/64 0.01 0.98

The rabbit total IgGs, containing anti-asparaginase IgGs, interact withL-asparaginase starting from the dilution 1/64 and cause totalinhibition of the enzymatic activity (99.02% of enzyme precipitated).

Example 5 Inactivation, by the Rabbit Total IgGs, of Free L-AsparaginaseInjected in the Mouse

An experiment was set up for the mouse (16 mice) to investigate theinhibition of L-asparaginase by the anti-asparaginase IgGs in vivo.

The experimental conditions were as follows:

-   -   dose of 100 IU/kg of L-asparaginase, equivalent to 1.25 IU/ml        circulating in a 25 g mouse    -   injection of 7.5 μg of rabbit total IgGs.

Injection of the IgGs or of PBS is carried out 20 minutes beforeinjection of L-asparaginase, free or encapsulated in mouse red bloodcells (Asp-RBC). Then 6 hours after this last-mentioned injection, themice are sacrificed and the blood is collected.

The L-asparaginase activity is then assayed in the plasma and in theRBCs. Table 4 summarizes the values obtained:

TABLE 4 L-asparaginase activity (IU/ml) IgGs PBS Asp-RBC Red blood cells0.798 ± 0.126 0.879 ± 0.146 Plasma 0.013 ± 0.002 0.126 ± 0.029 Free L-Red blood cells 0.132 ± 0.019 0.098 ± 0.013 asparaginase Plasma 0.002 ±0.002 0.417 ± 0.103

Assay of L-asparaginase in the RBCs of mice that received the Asp-RBCsdetects 0.798 and 0.879 IU/ml in the presence of IgGs or of PBSrespectively. Therefore the IgGs present in the plasma did not have aninhibitory effect on the encapsulated enzyme. In the plasma of thesesame mice, free L-asparaginase injected with the Asp-RBCs (there isstill in fact a small amount of free enzyme outside of the RBCs, of theorder of 10% of the dose) is inhibited in the presence of the IgGs(0.013 IU/ml) and remains active in the presence of PBS (0.126 IU/ml).

When free L-asparaginase was injected, the IgGs inhibited its activity(0.002 IU/ml) whereas injection of PBS had no effect on the activity ofthe enzyme (0.417 IU/ml). Taking into account the half-life of freeL-asparaginase (10 hours), the measurement of 0.417 IU/ml of plasmaL-asparaginase corresponds to the residual activity of the free enzyme 6hours after its injection.

The plasma concentration of L-asparagine was measured in the plasma of14 of the mice in the study (two could not be used as the volume was toosmall). The results are presented in Table 5.

TABLE 5 Plasma L-asparagine (μmol/liter) IgGs PBS Asp-RBC <2 <2 FreeL-asparaginase 28.09 ± 3.63 <2

The depletion of L-asparagine was total when the mice were treated withAsp-RBCs or when they received free L-asparaginase in the presence ofPBS.

Only the mice treated with free L-asparaginase in the presence of IgGhave a plasma concentration of L-Asparagine of 28.09 μM. The enzyme wastherefore inhibited in the plasma by the IgGs.

Example 6 Measurement of the Inhibition of Rabbit Total IgGs on theEnzymatic Activity of Peg-Asparaginase

Rabbit total IgGs, containing anti-asparaginase IgGs, were tested onPEG-asparaginase (Sigma # A5336). The same experiment as that presentedin Example 4 was carried out with the PEG-asparaginase. The results arepresented in Table 6.

TABLE 6 PEG-Aspa PEG-Aspa Residual Tube added Dilution assayed activityin No. (IU/ml) of the IgGs (IU/ml) supernatant (%) 1 0.40 — 0.360 90.002 0.79 1/8  0.023 2.97 3 0.79 1/16 0.026 3.32 4 0.79 1/32 0.023 2.90 50.79 1/64 0.029 3.67

The rabbit IgGs containing anti-asparaginase IgGs interacted with thePEG-asparaginase. The activity detected in the supernatant is less than4% of the initial mixed activity with the IgGs.

Example 7 Test Protocol

This protocol applies to a patient who is being considered for treatmentwith a particular asparaginase. A small blood sample is taken from thispatient and is treated conventionally to obtain a serum sample.

Then the following procedure is followed:

-   (a₀) optionally inactivation or removal of any asparaginase present    in the serum sample, or measurement of the residual enzymatic    activity-   (a) incubation of the sample with 0.1 to 5 IU/ml of asparaginase for    1 to 60 min;-   (a₁) optionally removal of the immune complexes, preferably by    centrifugation at 3000-25000 g for 1 to 30 min at the specified    speed;-   (b) incubation of the mixture from (a) or of the supernatant from    (a₁) for 2 to 60 min with a saturating amount of asparagine, notably    from 10 to 50 mg/ml;-   (c) incubation, notably for 3 to 20 min, of the preceding mixture    with the reagent system (α-ketoglutarate, NADPH, glutamate    dehydrogenase) that is able to detect the residual enzymatic    activity;-   (d) qualitative or quantitative evaluation of the loss or retention    of enzymatic activity, which correlates with the presence or the    content of asparaginase neutralizing factors in the sample.

It is possible to determine levels or thresholds of levels of residualenzymatic activity depending on whether or not stage (a₁) is included.

In a patient who has already been treated, if the method shows markedpresence of neutralizing factors, a replacement treatment using adifferent form of asparaginase is recommended and applied. Notably, whenthe free or modified form is likely to be inhibited, the enzymeencapsulated in erythrocytes is recommended.

The same protocol is applicable simply for testing the potentialefficacy of an asparaginase in the patient.

Example 8 Effect of Patient Serum Matrix on L-Asparaginase ActivityMeasurement

Three human sera, naïve to L-asparaginase treatment, were mixed with 7L-asparaginase concentrations (from 0 to 800 IU/L) to ensure that humanserum has no interference on L-asparaginase activity measurement.Samples were incubated 15 minutes at 37° C. and were centrifuged 10minutes at 17500 g at 4° C. L-asparaginase activity is checked in thesupernatant. As a control, buffer 1×PBS 4% BSA was mixed with the sameL-asparaginase concentrations. Results are presented in Table 7.

TABLE 7 Added L- asparaginase Measured L-asparaginase activity (IU/L)condition IU/L sérum 246922 sérum 25579 sérum 255810 pure 800 685 783788 serum 400 ND* 442 461 200 220 220 246 100 101 125 122 50 30 76 72 2519 37 33 0 0 0 3 buffer 800 753 802 851 control 400 407 455 463 200 218236 244 100 115 120 124 50 63 59 77 25 24 45 37 0 0 — 0 *Not Determined

No interference of human serum was observed on L-asparaginase activitymeasurement (compared with buffer control: see Table 7 and FIG. 1).

Example 9 Effect of Patient Serum Matrix on L-Asparaginase ActivityMeasurement and Determination of Basal Activity of Human Serum

Fifty-two human sera, naïve to L-asparaginase treatment, were mixed withL-asparaginase at a final concentration of 500 IU/L to ensure that humanserum has no interference on L-asparaginase activity measurement.Samples were incubated 15 minutes at 37° C. and were centrifuged 10minutes at 17500 g at 4° C. L-asparaginase activity is checked in thesupernatant. As a control, buffer 1×PBS 4% BSA was mixed with the sameL-asparaginase final concentration. Results are presented in Table 8 andin FIG. 2.

TABLE 8 Measurement of I-asparaginase activity on 52 human sera withL-asparaginase added at a final concentration of 500 (IU/L) SerumMeasured L-asparaginase activity (IU/L) 1 467 2 493 3 505 4 492 5 466 6482 7 470 8 474 9 453 10 474 11 482 12 472 13 467 14 469 15 468 16 45617 476 18 477 19 481 20 477 21 479 22 486 23 478 24 479 25 472 26 479 27457 28 457 29 483 30 472 31 418 32 462 33 477 34 477 35 467 36 442 37464 38 464 39 489 40 452 41 475 42 484 43 480 44 480 45 475 46 478 47473 48 482 49 480 50 473 51 478 52 477 Control 480 Mean 473.02 Standarddeviation 13.4 Mean − 2SD 446.22 Mean + 2SD 499.82

The mean activity measured for the 52 human sera is 473 IU/L thestandard deviation (SD) is 13.4 IU/L. The mean activity measured for thesera is not significantly different from the control activity. Allvalues are comprised within an acceptable range: on the 52 measurementsonly 3 are outside the confidence range of [mean−2SD; mean+2SD]. Thedistribution of the activity measured according to the serum assayedindicate that L-asparaginase activity is not affected by the matrixserum (FIG. 2).

To check the absence of enzymatic activity signal in human serum: 25human sera naïve to L-asparaginase treatment were assayed for1-asparaginase activity.

TABLE 9 Measurement of l-asparaginase activity on 25 human sera naïve toL-asparaginase treatment Serum Measured L-asparaginase activity (IU/L) 10 2 2 3 2 4 1 5 2 6 0 7 0 8 0 9 0 10 0 11 0 12 0 13 0 14 1 15 2 16 1 175 18 2 19 0 20 3 21 1 22 0 23 0 24 0 25 0 Mean 0.88 Standard Deviation1.27

With each of the 25 human sera assayed the L-asparaginase activity isclosed to zero. The mean activity measured is 0.88 IU/L and the standarddeviation is 1.27 IU/L. The maximum activity measured is 5 IU/L forserum 17 this basal activity signal is not likely to affect themeasurement of L-asparaginase activity as it represents 1% of theactivity measured for sera mixed with L-asparaginase at a finalconcentration of 500 IU/L.

Example 10 Inhibition of L-Asparaginase Activity by Anti-AsparaginaseIgG Spiked Human Sera

Two human sera, naïve to L-asparaginase treatment, were pooled andspiked with anti-asparaginase IgG concentrations ranging between 1 and100 μg/mL (1, 2, 5, 10, 20, 40, 80, 100 μg/mL). The anti-asparaginaseIgG were obtained as described in examples 1 and 2.

Then 500 IU/L of L-asparaginase was added. Samples were incubated 15minutes at 37° C. and were centrifuged 10 minutes at 17500 g at 4° C.Residual L-asparaginase activity was measured in the supernatant. As acontrol, buffer 1×PBS 4% BSA was used instead of the human sera pool.Results are presented in Table 10 and FIG. 3.

TABLE 10 Added L- measured L- Added Added anti- asparagi- asparaginaseinhibi- control IgG aspa IgG nase activity tion (μg/mL) (μg/mL) (IU/L)(IU/L) (%) human 100 500 589 0.00 serum 40 500 537 0.00 pool 0 0 — 0 500534 0.00 1 500 518 3.00 2 500 548 0.00 5 500 515 3.56 10 500 426 20.2220 500 4 99.25 40 500 3 99.44 80 500 1 99.81 100 500 0 100.00 buffer 100500 558 0.00 control 40 500 592 0.00 0 0 — 0 500 564 0.00 1 500 595 0.002 500 562 0.00 5 500 550 2.48 10 500 378 32.98 20 500 7 98.76 40 500 ND*— 80 500 2 99.65 100 500 0 100.00 *Not Determined

When L-asparaginase is mixed with increasing concentrations ofanti-asparaginase IgG (specific IgG), in human serum or buffer control,total enzymatic activity inhibition occurs at an IgG concentration of 20μg/mL and higher. Partial inhibition occurs when enzyme is mixed with 5to 20 μg/mL anti-asparaginase IgG. Below 5 μg/mL anti-asparaginase IgG,L-asparaginase activity is not inhibited.

No inhibition is observed when L-asparaginase is incubated withnonspecific IgG (see added control IgG in Table 10).

To refine the inhibition reaction of anti-asparaginase IgG concentrationbetween 10 and 20 μg/mL on L-asparaginase activity, an experiment wasperformed with IgG concentrations ranging from 8 to 22 μg/mL. As usual,L-asparaginase is added to a final concentration of 500 IU/L. All thesamples were incubated 15 minutes at 37° C. and were centrifuged 10minutes at 17500 g at 4° C. Residual L-asparaginase activity is measuredin the supernatant. The assay is performed with a human serum pool.Results are presented in Table 11 and FIG. 4.

TABLE 11 Added measured Added anti-aspa L-asparaginase L-asparaginaseinhibition IgG (μg/mL) (IU/L) activity (IU/L) (%) human 2 — serum 22 1 —pool 500 580 — 8 500 510 12.07 10 500 410 29.31 12 500 98 83.10 14 50065 88.79 16 500 5 99.14 18 500 1 99.83 20 500 0 100.00 22 500 2 99.66

Total inhibition of L-asparaginase activity appears at an IgGconcentration of 16 μg/mL. Below 16 μg/mL, L-asparaginase activityinhibition is partial.

The opposite reaction was tested: a fixed anti-asparaginase IgGconcentration (13.64 μg/mL corresponding to 80% inhibition) was mixedwith L-asparaginase concentrations ranging from 500 to 10000 IU/L.Samples were incubated 15 minutes at 37° C. and were centrifuged 10minutes at 17500 g at 4° C. Residual L-asparaginase activity is measuredin the supernatant. Results are shown in Table 12 and FIG. 5.

TABLE 12 Added measured Added anti-aspa L-asparaginase L-asparaginaseinhibition IgG (μg/mL) (IU/L) activity (IU/L) (%) human 13.64 0 — serum13.64 500 36 93.00 pool 13.64 1000 716 32.00 13.64 5000 5010 9.00 13.6410000 10780 3.00 500 548 — 1000 1052 — 5000 5480 — 10000 11080 —

The more concentrated L-asparaginase is, the less the fixed IgGconcentration (13.64 μg/mL) inhibits its activity. A fixed quantity ofanti-asparaginase IgG inhibits a fixed quantity of L-asparaginase.Therefore, L-asparaginase activity inhibition is dose-dependent.

Example 11 Test of 57 Human Sera from 17 Patients Treated withL-Asparaginase

To ensure the assay has the ability to quantify the neutralization ofasparaginase in a patient: 57 human sera sampled from 17 Acutelymphoblastic leukaemia patients under treatment with L-asparaginasewere tested according to the test protocol described in example 7. Thesamples were taken at different time of treatment course and ameasurement of residual L-asparaginase enzyme activity was conducted toverify that this activity is negligible and will not interfere with thetest procedure.

The sera were then mixed with L-asparaginase at a final concentration of500 IU/l and were incubated 15 minutes at room temperature. TheL-asparaginase activity was then determined before and after acentrifugation step of 6 minutes at 7800 rpm. As a control, buffer 1×PBS4% BSA was mixed with L-asparaginase at a final concentration of 500IU/L. Results are presented in the table 13 below and in FIG. 6.

TABLE 13 Test of 57 human sera from 17 patients under treatment withL-asparaginase Activity after Activity after % of % of Initial additionof L- addition of L- inhibition inhibition activity asparaginaseasparaginase and without after Patient Serum (IU/L) (IU/L)centrifugation (IU/L) centrifugation centrifugation Control 6 655 662 NANA 1 1 2 551 515 15.88 22.21 2 2 440 342 32.82 48.34 3 1 559 518 14.6621.75 4 2 658 655 −0.46 1.06 2 5 7 NA 441 NA 33.38 6 13 767 727 −17.1−9.82 3 7 1 706 701 −7.79 −5.89 8 −2 724 716 −10.53 −8.16 9 11 692 731−5.65 −10.42 10 2 680 666 −3.82 −0.6 11 1 711 733 −8.55 −10.73 12 1 754711 −15.11 −7.4 13 2 726 730 −10.84 −10.27 4 14 2 664 744 −1.37 −12.3915 15 804 667 −22.75 −0.76 Control 6 636 NA NA 5 16 1 690 516 −8.4918.87 17 2 649 678 −2.04 −6.6 18 2 684 575 −7.55 9.59 19 ND 622 633 2.20.47 20 1 670 604 −5.35 5.03 21 1 609 583 4.25 8.33 6 22 1 659 519 −3.6218.4 7 23 2 508 492 20.13 22.64 8 24 1 761 473 −19.65 25.63 25 0 633 6080.47 4.4 9 26 4 705 524 −10.85 17.61 27 2 656 525 −3.14 17.45 10 28 3608 623 4.4 2.04 29 1 649 560 −2.04 11.95 30 3 610 609 4.09 4.25 31 7694 570 −9.12 10.38 32 −1 598 586 5.97 7.86 33 2 589 516 7.39 18.87 34 0527 NA 17.14 NA Control 3 661 629 NA NA 11 35 2 713 587 −7.87 6.68 36 1499 667 24.51 −6.04 37 3 596 666 9.83 −5.88 38 2 701 579 −6.05 7.95 39 2850 408 −28.59 35.14 12 40 2 798 446 −20.73 29.09 41 2 599 596 9.38 5.2542 5 814 448 −23.15 28.78 43 3 752 265 −13.77 57.87 13 44 1 705 570−6.66 9.38 14 45 1 685 659 −3.63 −4.77 15 46 1 573 388 13.31 38.31 47 2492 408 25.57 35.14 48 1 605 678 8.47 −7.79 49 2 571 355 13.62 43.56 502 725 275 −9.68 56.28 51 2 464 437 29.8 30.52 52 3 497 475 24.81 24.4853 0 540 445 18.31 29.25 16 54 3 550 524 16.79 16.69 55 2 568 637 14.07−1.27 56 1 595 379 9.98 39.75 17 57 1 709 585 −7.26 7 ND: Not determinedNA: Not Applicable

All the samples have a residual asparaginase activity which isnegligible, the highest residual activity is of 15 IU/L and should notinterfere with the test procedure as it represents only 3% of thetheoretical added L-asparaginase. The asparaginase activity measured forthe control is higher than expected (655, 636 and 661 IU/L respectivelyfor the 3 control compared to the 500 IU/L that were expected). Thepercentage of inhibition of asparaginase activity was calculated basedon enzymatic activity measured for the control. The fact that numerousvalues of inhibition percentage are negative indicate a bias in themeasurement procedure.

The percentage of inhibition of asparaginase activity is higher aftercentrifugation suggesting that the centrifugation step has eliminatedsome immune complexes that were formed between asparaginase andanti-asparaginase antibodies.

On the 17 patients assayed 14 experience an inhibition of theasparaginase activity by factors present in their serum (FIG. 6). Heightpatients have a percentage of inhibition above 20% but only threepatients have a percentage of inhibition higher than 40% (FIG. 6).

The invention claimed is:
 1. An in-vitro method for assaying a patient'scompatibility to asparaginase therapy comprising: (a) obtaining a sampleof blood, plasma, or serum from a patient; (b) incubating said samplewith a known amount of asparaginase for a period of time sufficient toproduce antibody-asparaginase immune complexes; (c) removing from theincubated sample any antibody-asparaginase immune complexes formedduring incubation step (b) and recovering a sample free ofantibody-asparaginase immune complexes; (d) incubating the sampleobtained at step (c) with asparagine, (e) determining a residualasparaginase activity or the amount of asparaginase residual activity inthe resultant mixture of step (d).
 2. The method according to claim 1,further comprising determination or quantification of the presence ofneutralizing factors.
 3. The method according to claim 1, wherein thepatient is currently being treated with asparaginase or has been treatedwith asparaginase.
 4. The method according to claim 1, wherein measuringthe activity of the asparaginase in the mixture is carried out byadding, to the mixture, asparagine and a reagent system suitable forassaying residual enzymatic activity.
 5. The method according to claim4, comprising the following stages: (a) incubation of the mixture withthe reagent system suitable for assaying the residual enzymaticactivity; and (b) qualitative or quantitative evaluation of a loss orretention of enzymatic activity, which is correlated with the presenceor with the content of neutralizing factors of said asparaginase in thesample.
 6. The method according to claim 1, comprising, beforeincubating the sample with said form of asparaginase, a stage (a_(o)) ofremoval or inactivation of any asparaginase that may be present in thesample.
 7. The method according to claim 1, comprising, beforeincubating the sample with said form of asparaginase, a stage (a_(O)) ofmeasurement of the baseline content of asparaginase in the sample. 8.The method according to claim 4, wherein the reagent system is sensitiveto the appearance of ammonium ions resulting from enzymatic degradationof asparagine by asparaginase.
 9. The method according to claim 8, whichemploys a reaction that consumes the ammoniums ions quantitatively. 10.The method according to claim 9, wherein the quantitative consumption ofthe ammonium ion is measured by measuring the decrease in opticaldensity of the mixture.
 11. The method according to claim 4, whichemploys the following reactions:asparaginase+asparagine aspartic acid+NH4⁺  (1)α-ketoglutarate+NH4⁺+NADPH+glutamate dehydrogenase (catalyst)glutamate+NADP⁺+H₂O  (2).
 12. The method according to claim 1, whichcomprises determination of the patient's capacity to respond: (i)positively to treatment with the asparaginase; (ii) not respond to it;or (iii) only respond incompletely.
 13. The method according to claim12, which comprises, for cases (ii) and (iii), treating the patientusing a different asparaginase.
 14. The method according to claim 12,which comprises, for case (i), treating the patient using theasparaginase.
 15. The method according to claim 13, wherein thedifferent asparaginase is an asparaginase included in a biovector. 16.The method of claim 1, further comprising identifying the asparaginaseis active in the patient sample and is able to be effective in thepatient if the activity is present.
 17. The method of claim 1, wherein amedium is derived from the sample obtained in step (a) and used in theincubation step (b).